Full-length ISP3 was amplified by PCR using primers OL1459 and OL1320

Full-length ISP3 was amplified by PCR using primers OL1459 and OL1320. from the lack of serine peptidase inhibition during promastigote uptake. isp2/3 mutants were more infective than wild-type parasites to BALB/c mice at the early stages of infection, but became Pralatrexate equivalent as the infection progressed. These data support the hypothesis that ISPs ofL. majortarget host serine peptidases and influence the early stages of infection of the mammalian host. == Introduction == Leishmaniaare trypanosomatid parasitic protozoa that cause a spectrum of human diseases, the leishmaniases, ranging from a lethal visceral infection to milder cutaneous ulcers. Their life cycle involves a flagellated procyclic promastigote stage that multiplies in the sandfly gut, a non-dividing flagellated metacyclic promastigote stage within the sandfly mouth parts, and a non-motile amastigote form that proliferates in mammalian macrophages. Peptidases play important roles in the pathogenicity of many parasitic protozoa, includingLeishmania(Sajid and McKerrow, 2002;Mottramet al., 2004). The genome ofLeishmania major, one of the causative agents of cutaneous leishmaniasis in the Old World, has been sequenced (Ivenset al., 2005), revealing the presence of at least 153 identifiable peptidases that can be classified into the major catalytic types FOS (cysteine-, serine-, metallo-, threonine and aspartic-) and represent 2% of the genome. One of the major mechanisms used to control the activity of peptidases in mammals is a tight interaction with natural peptidase inhibitors such as serpins, cystatins and tissue inhibitor of metalloproteinases (Rawlingset al., 2004). TheL. majorgenome does not appear to contain any orthologues of these molecules, yet it is not devoid of natural peptidase inhibitors (Ivenset al., 2005). The first to be identified inL. majorwas an inhibitor of cysteine peptidases (ICP), which is Pralatrexate a member of the chagasin family of inhibitors first identified inTrypanosoma cruzi(Monteiroet al., 2001) and subsequently found in a variety of bacterial and protozoan pathogens (Rigdenet al., 2002;Sandersonet al., 2003). InT. cruziandTrypanosoma bruceichagasin/ICP is a modulator of parasite differentiation (Santoset al., 2005;2007), whileL. majorICP is thought to play a role in the hostparasite interaction (Besteiroet al., 2004).L. majorICP andT. cruzichagasin have an unusual immunoglobulin-like fold with a cystatin-like mechanism of inhibition, which distinguishes them from all other known peptidase inhibitors (Salmonet al., 2006;Smithet al., 2006). A second group of putative peptidase inhibitors identified in theL. majorgenome are orthologues of theEscherichia coliserine peptidase (SP) inhibitor ecotin and have been termedinhibitor ofserinepeptidases (ISPs). Ecotin is an 18 Pralatrexate kDa protein first isolated from the periplasm ofE. coli(Chunget al., 1983). It forms dimers and inhibits a wide range of SPs from the S1A peptidase family (trypsin fold) of clan PA(S), which includes trypsin, chymotrypsin, neutrophil elastase (NE) and cathepsin G (Chunget al., 1983;McGrathet al., 1995). There are no reports that ecotin inhibits SPs of other families or catalytic classes. In addition,Chunget al. (1983)were unable to identify anE. colipeptidase sensitive to ecotin, suggesting that ecotin may protect the cell against exogenous S1A peptidases (Eggerset al., 2004), which are involved in processes such as coagulation, fibrinolysis and host defence (Stoop and Craik, 2003). The structure of ecotin and its mechanism of SP inhibition have been investigated extensively (McGrathet al., 1991a;Perona and Craik, 1997;Yanget al., 1998). Ecotin has also been studied as a macromolecular scaffold to develop specific peptidase inhibitors with potential therapeutic applications (Stoop and Craik, 2003). Methionine is present in its reactive site (McGrathet al., 1991b), although it is not essential for ecotin’s inhibitory activity against trypsin (Seonget al., 1994). This dichotomy was resolved when the crystal structure of ecotin revealed that the protein forms a head to tail dimer that interacts with its substrate via multiple binding sites, including the active site of the peptidase (Shinet al., 1996;Yanget al., 1998). This also explains why a wide range of SPs from the S1A family are inhibited by ecotin. AlthoughL. majorhas 13 SPs belonging to six families, the parasite apparently lacks genes encoding SPs from the S1A family of clan PA(S) (Ivenset al., 2005). Three ecotin orthologous ISP genes are present in theL. majorgenome and while it is.